Part:BBa_K2332021
Blue light repressible bioluminescence (Pblrep LuxCDABE )
Our LIT Bulb will be composed of light sensitive E. coli that can detect sunlight (blue light) to repress bioluminescence (LuxCDABE). In the presence of light transcription of the LuxCDABE for the production of Luciferase and substrates will be inhibited. In the dark, the inhibition will be released and bioluminescence will be induced. This part encodes the necessary requirements for bioluminescence production, codon optimized for E. coli.
This part encodes the production of the proteins required for bacterial luminescence including luciferase and its substrates from Vibrio fischeri, codon optimised for E. coli under the control of our blue light repressible promoter, (Pblrep). Naturally, Vibrio fischeri use additional regulatory proteins (encoded by LuxR and LuxI upstream) whose external concentration increases as a function of increasing cell-population density. Bacteria detect the accumulation of a minimal threshold stimulatory concentration of these autoinducers and regulate the expression of LuxCDABE. Consequently, we removed these regulatory proteins to ensure that cell concentration dependent luminescence through quorum sensing is surpassed and our cultures can fluoresce independently. Additionally, by using our blue light repressible promoter (PBLrep), we will ensure that LuxCDABE expression is only active in the dark and excess buildup of substrates and luciferase, potentially toxic, will be prevented.
The blue light repressible promoter (PBLrep) consists of the 18bp DNA binding region of EL222, a natural photosensitive DNA-binding protein from the marine bacterium Erythrobacter litoralis HTCC2594, positioned between the -35 and -10 regions of the RNAP binding site [1]. In the dark, EL222 is inactive as its N-terminal LOV domain represses its DNA-binding C-terminal HTH domain. In the daytime, exposure to blue light (450nm) results in the LOV-HTH interaction to be released, allowing it to dimerize and bind its binding region, causing steric hindrance to RNAP binding, ultimately repressing transcription. Therefore, only at nighttime the transcription of LuxCDABE will occur.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2621
Illegal AgeI site found at 2888
Illegal AgeI site found at 3416
Illegal AgeI site found at 3997
Illegal AgeI site found at 4444
Illegal AgeI site found at 5322
Illegal AgeI site found at 5537 - 1000COMPATIBLE WITH RFC[1000]
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